This application addresses Broad Challenge Area 04, "Clinical Research" and two specific Challenge Topics: 03-DK-101, "Discovery of biomarkers for disease risk, progression or response to therapy in diseases of interest of NIDDK;" and 04-DK-102, "Improved techniques for clinical diagnosis, detailed clinical phenotyping, and clinical disease staging and activity for conditions of interest to NIDDK." The goal of this project is to identify objective, quantitative bio-molecular parameters that correlate with healing outcomes for diabetic foot ulcers (DFUs). Chronic non-healing DFUs are a widespread and serious clinical problem with high rates of morbidity, disability and mortality, including a high risk for lower limb amputations. About half of all DFUs fail to heal promptly or completely in response to the current standard of care. One of the major obstacles to prompt and complete DFU healing is the inability to predict which wounds are not likely to respond to standard treatment protocols and will require alternative interventions. The edges of chronic wounds contain tissue that expresses inhibitors of healing and does not respond to wound-healing stimuli. The challenge of this project is to develop a feasible quantitative method of distinguishing between tissue that has the capacity to heal and tissue that does not, thus identifying non-healing phenotype. This project proposes to identify biomarkers that can objectively and quantifiably identify non-healing tissue in DFUs. Two protein markers -- nuclear localization of [unreadable]-catenin and induced expression of c-myc -- have been associated with healing impairment in other chronic wounds but have not been studied in DFUs. Therefore, the objective of this project is to determine whether expression of [unreadable]-catenin and/or c-myc at the wound edge is correlated with objective quantitative measures of DFU wound closure. Fifty consecutive patients receiving the current standard of care for chronic DFUs will provide small wound tissue biopsies at the initial treatment (week 0) and 4 weeks later. The extent of wound closure at 4 weeks will be used as a surrogate outcome for wound healing and will be confirmed by histopathology. Aim 1 is to determine whether [unreadable]-catenin localization in the nucleus is correlated with non-healing. [unreadable]-catenin localization will be quantified by immunohistochemistry. The primary outcome measure will be the correlation between the extent of wound closure at week 4 and nuclear [unreadable]-catenin staining. Aim 2 is to determine whether c-myc protein expression is correlated with non-healing. c-myc expression will be quantified by immunohistochemistry. The primary outcome measure will be the correlation between extent of wound closure and nuclear [unreadable]-catenin staining. The secondary outcome measure will be the correlation between wound closure and expression of both markers together. This project is ideally suited to the goals of the NIH Challenge Initiative and the selected Challenge Topics because it advances the science of wound healing by identifying objective cellular biomarkers that predict healing outcomes and provides improved quantitative techniques that can be used to monitor treatment efficacy and clinical outcomes. This project has a high likelihood of achieving important milestones within two years because it is supported by an established and productive team of collaborators that has existing Institutional Review Board (IRB) approvals for DFU biopsy and analysis and has all of the methods and protocols validated and fully described in prior publications. Funds from this grant will create full-time and part- time jobs and support students to clinical and translational research. The long-term goal of this research program is to reduce the number of amputations and improve healing outcomes for millions of individuals suffering from the consequences of chronic non-healing skin ulcers. Successful completion of this project will result in identification of reliable biological markers that can be used to identify non-healing wound tissue and predict which diabetic foot ulcers are incapable of healing without timely and aggressive intervention.